Interaction of Ethanol and Oral ANS-6637, a Selective ALDH2 Inhibitor in Males: A Randomized, Double-Blind, Placebo-Controlled, Single-Ascending Dose Cohort Study.

BACKGROUND: ANS-6637, an orally bioavailable selective and reversible aldehyde dehydrogenase-2 (ALDH2) inhibitor, is under development for drug and alcohol use disorders. During the elimination of alcohol, ALDH2 metabolizes acetaldehyde to acetate; inhibiting this enzyme can lead to aversive reactions due to the accumulation of acetaldehyde. Thus, understanding the safety and tolerability of ANS-6637 in combination with alcohol is essential. TRIAL DESIGN AND METHODS: Forty eight healthy males participated in a randomized, double-blind, placebo-controlled, single-ascending dose cohort study of oral ANS-6637. Eligible participants were randomized to ANS-6637 (n = 36) or placebo (n = 12) in a 3:1 fashion in each of 6 dose cohorts (8 per cohort; ANS-6637 dose levels were 25, 50, 100, 200, 400, and 600 mg). Two hours after receiving study drug, participants drank up to 5 standard drinks, 1 every 30 minutes. Safety assessments, pharmacodynamic measures, and pharmacokinetic blood samples were obtained. RESULTS: Flushing was the most common adverse event (AE) associated with ANS-6637 (24 of 36 participants) compared with placebo (3 of 12). Statistically significant, but modest, increases in heart rate (HR) occurred (+10.5 bpm after 2 drinks; +16.9 to + 20.5 bpm after 3rd through 5th drink). No participant met HR or systolic blood pressure criteria for stopping ethanol administration. There were no clinically significant QTc interval prolongations. Individuals receiving ANS-6637 reported lower ratings of liking, alcohol effects, and feeling drunk. CONCLUSIONS: A single oral dose of ANS-6637 with up to 5 standards drinks over 2.5 hours was generally well tolerated in healthy males. The most common pharmacological response was flushing and an increase in HR, which are known effects of acetaldehyde accumulation and consistent with inhibition of ALDH2 with oral ANS-6637 in combination with alcohol. The results of this alcohol interaction study support further testing of ANS-6637 in individuals who consume alcohol heavily.

Case Discussions and Radiographic Illustration of Magnet-Related Injuries in Children.

BACKGROUND: Injuries from multiple magnet ingestions in the pediatric population have been increasing in both incidence and morbidity. This trend will likely continue after a 2017 court ruling that overturned a ban on the sale of magnet sets marketed as "adult desk toys." Depending on the arrangement of the ingested magnets in the gastrointestinal tract, the consequences can range from benign to life threatening. OBJECTIVE: This review of cases aims to help clinicians recognize this pathology and help them appreciate the unique management of this type of foreign body ingestion. DISCUSSION: Several cases are presented that individually illustrate an arm of the most comprehensive management algorithm, proposed by the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition. The management is largely driven by the clinical appearance of the child as well as information obtained through abdominal radiographs. Imaging variables that factor into management include the location of the magnets, the number of magnets, and the progression of magnets on serial radiographs. CONCLUSION: This article uses cases and illustrative medical imaging to describe the most common scenarios and their management. This is especially relevant considering recent U.S. court rulings that overturned the U.S. Consumer Product Safety Commission's ban on the sale of toys containing multiple miniature magnets.

Alternative designs for clinical trials in rare diseases.

Evidence-based medicine requires strong scientific evidence upon which to base treatment. In rare diseases, study populations are often small, and thus this evidence is difficult to accrue. Investigators, though, should be creative and develop a flexible toolkit of methods to deal with the problems inherent in the study of rare disease. This narrative review presents alternative clinical trial designs for studying treatments of rare diseases, including cross-over and n-of-1 trials, randomized placebo-phase design, enriched enrollment, randomized withdrawal design, and classes of adaptive designs. Examples of applications of these designs are presented along with their advantages and disadvantages. Additional analytical considerations such as Bayesian analysis, internal pilots, and use of biomarkers as surrogate outcomes are further discussed. A framework for selecting appropriate clinical trial design is proposed to guide investigators in the process of selecting alternative designs for rare diseases. © 2016 Wiley Periodicals, Inc.

GS 455534 selectively suppresses binge eating of palatable food and attenuates dopamine release in the accumbens of sugar-bingeing rats.

Binge eating palatable foods has been shown to have behavioral and neurochemical similarities to drug addiction. GS 455534 is a highly selective reversible aldehyde dehydrogenase 2 inhibitor that has been shown to reduce alcohol and cocaine intake in rats. Given the overlaps between binge eating and drug abuse, we examined the effects of GS 455534 on binge eating and subsequent dopamine release. Sprague-Dawley rats were maintained on a sugar (experiment 1) or fat (experiment 2) binge eating diet. After 25 days, GS 455534 was administered at 7.5 and 15 mg/kg by an intraperitoneal injection, and food intake was monitored. In experiment 3, rats with cannulae aimed at the nucleus accumbens shell were maintained on the binge sugar diet for 25 days. Microdialysis was performed, during which GS 455534 15 mg/kg was administered, and sugar was available. Dialysate samples were analyzed to determine extracellular levels of dopamine. In experiment 1, GS 455534 selectively decreased sugar intake food was made available in the Binge Sugar group but not the Ad libitum Sugar group, with no effect on chow intake. In experiment 2, GS 455534 decreased fat intake in the Binge Fat group, but not the Ad libitum Fat group, however, it also reduced chow intake. In experiment 3, GS 455534 attenuated accumbens dopamine release by almost 50% in binge eating rats compared with the vehicle injection. The findings suggest that selective reversible aldehyde dehydrogenase 2 inhibitors may have the therapeutic potential to reduce binge eating of palatable foods in clinical populations.

Ranolazine attenuates mechanical allodynia associated with demyelination injury.

OBJECTIVE: The aim of this study was to determine whether ranolazine, a new medication that targets sodium channels to improve cardiac ischemia and angina, could be an effective analgesic agent for pain associated with demyelination injury. BACKGROUND: Many agents have been used to treat neuropathic pain but not all neuropathic conditions respond similarly to treatment. We have demonstrated that ranolazine, an agent that blocks voltage-gated sodium channels Nav 1.4, 1.5, 1.7, and 1.8, is effective in attenuating mechanical hyperalgesia in both complete Freund's adjuvant and spared nerve injury preclinical models of inflammatory and neuropathic pain, respectively. Here we test the efficacy of this drug in a newly validated model of demyelination injury that responds uniquely to a number of treatment options. METHODS: After determination of baseline nerve conduction velocities (NCVs) and withdrawal responses from heat and mechanical stimulation in male Sprague-Dawley rats (300-350 g), 1 µg/30 µL of doxorubicin was injected into one sciatic nerve. The contralateral nerve provided a sham-injected control. Two weeks after doxorubicin injection, NCV and sensitivity to heat and mechanical stimulation were reassessed before and after treatment with ranolazine (10, 30, 50 mg/kg) administered intraperitoneally using an experimenter-blinded, randomized design. RESULTS: Doxorubicin injection produced a significant hyperalgesic effect in response to mechanical but not heat stimulation. Conduction velocities in the injected limbs were reduced when compared with controls. Ranolazine reduced mechanical allodynia with peak efficacy at 30 mg/kg. Fifty milligram/kilogram ranolazine restored NCVs by approximately 50%, but had no effect in the uninjected limb. CONCLUSIONS: Ranolazine exerts broad-spectrum actions to reduce mechanical allodynia that is associated with peripheral demyelination injury.

Designing clinical trials to address alcohol use and alcohol-associated liver disease: an expert panel Consensus Statement.

Most patients with alcohol-associated liver disease (ALD) engage in heavy drinking defined as 4 or more drinks per day (56 g) or 8 (112 g) or more drinks per week for women and 5 or more drinks per day (70 g) or 15 (210 g) or more drinks per week for men. Although abstinence from alcohol after diagnosis of ALD improves life expectancy and reduces the risk of decompensation of liver disease, few studies have evaluated whether treatment of alcohol use disorders will reduce progression of liver disease and improve liver-related outcomes. In November 2021, the National Institute of Alcohol Abuse and Alcoholism commissioned a task force that included hepatologists, addiction medicine specialists, statisticians, clinical trialists and members of regulatory agencies to develop recommendations for the design and conduct of clinical trials to evaluate the effect of alcohol use, particularly treatment to reduce or eliminate alcohol use in patients with ALD. The task force conducted extensive reviews of relevant literature on alcohol use disorders and ALD. Findings were presented at one in-person meeting and discussed over the next 16 months to develop the final recommendations. As few clinical trials directly address this topic, the 28 recommendations approved by all members of the task force represent a consensus of expert opinions.

Nucleus accumbens AGS3 expression drives ethanol seeking through G betagamma.

Approximately 90% of alcoholics relapse within 4 years, in part because of an enhanced motivation to seek alcohol (EtOH). A novel G protein modulator (Gpsm1/AGS3) was up-regulated in the rat nucleus accumbens core (NAcore) but not in other limbic nuclei during abstinence from operant EtOH self-administration. Furthermore, NAcore AGS3 knockdown reduced EtOH seeking to pre-abstinence levels in a novel rat model of compulsive, human EtOH seeking. AGS3 can both inhibit G protein G i alpha-mediated signaling and stimulate G betagamma-mediated signaling. Accordingly, sequestration of G betagamma, but not G i alpha knockdown, significantly reduced EtOH seeking to pre-abstinence levels. Thus, AGS3 and G betagamma are hypothesized to gate the uncontrolled motivation to seek EtOH during abstinence. AGS3 up-regulation during abstinence may be a key determinant of the transition from social consumption to compulsion-like seeking during relapse.

Graft type in pediatric liver transplantation.

PURPOSE OF REVIEW: To review the literature on graft type in pediatric liver transplantation, with a specific focus on publications since 2010. RECENT FINDINGS: Due to the limited availability of whole livers for transplantation, the majority of pediatric patients will receive a technical variant graft (live donor, reduced, split). Although the outcomes of these grafts may be inferior to whole organs, the detrimental impact needs to be balanced with the impact of technical variants on improved access and survival to transplantation. Vigilance in detecting and managing posttransplant complications is critical in ensuring the optimal outcome. Infants under 5 kg pose a particular challenge in terms of graft selection with hyperreduced and monosegment grafts proposed for this population. Grafts from donors after cardiac death show promise in expanding the donor pool. However, experience in pediatric patients with these grafts is quite limited, particularly in young children who form the majority of pediatric transplant recipients. Auxiliary transplantation is proposed as a strategy for children presenting with fulminant hepatic failure and for children with metabolic diseases. SUMMARY: The majority of children will receive a technical variant graft, with graft choice being largely determined by organ availability.

Outcomes after laparoscopic surgery in children with inflammatory bowel disease.

BACKGROUND: The utility and efficacy of the laparoscopic approach to the management of inflammatory bowel disease (IBD) in children are not clearly known. METHODS: We conducted a retrospective descriptive cohort study of children with a diagnosis of IBD who underwent a laparoscopic or laparoscopy-assisted procedure at a quaternary pediatric referral center between 1999 and 2007. RESULTS: One-hundred thirty-six children underwent 154 operations (85 small bowel/ileocolic and 69 colorectal) over the 8 years of the study. Median age was 14.8 years (range = 1.8-18.8). The diagnosis was Crohn's disease in 83, ulcerative colitis in 50, and indeterminate colitis in 3. Median time to regular diet was 5 days (range = 1-19), and median postoperative stay was 7 days (range = 1-70). Seven patients undergoing a small bowel/ileocolic resection (8.2%) were converted to an open procedure. Overall morbidity for the small bowel/ileocolic procedures was 27.1%. The conversion rate during subtotal colectomy (STC) was 7.1% (3/42), and it was 0% for the 22 patients who underwent ileal pouch-anal anastomosis (IPAA) procedures. Overall morbidity associated with STC was 62.8%, and following IPAA it was 63.6%. Sixteen percent (7/69) of those who underwent a colorectal procedure developed a late postoperative bowel obstruction with three patients requiring operative intervention. CONCLUSION: A laparoscopic approach is feasible with a low conversion rate in most children with IBD. Despite superior cosmesis, perioperative morbidity is similar to that seen with open procedures. Laparoscopic colorectal IBD procedures are associated with an unexpectedly high incidence of postoperative bowel obstruction, although the rates are comparable to those seen with open surgery.

Ranolazine attenuation of CFA-induced mechanical hyperalgesia.

OBJECTIVE: To determine whether ranolazine, a new anti-angina medication, could be an effective analgesic agent in complete Freund's adjuvant-induced inflammatory pain. BACKGROUND: Plantar injection of complete Freund's adjuvant (CFA) produces an extended period of hyperalgesia that is associated with a dramatic up-regulation of Na(v) 1.7 sodium channels in populations of large and small dorsal root ganglion neurons related to the injection site. Ranolazine appears to produce its anti-angina effect through blocking the late sodium current associated with the voltage-gated sodium channel, Na(v) 1.5. Because ranolazine also inhibits Na(v) 1.7, and 1.8, we sought to determine whether it could be an effective analgesic agent in CFA-induced inflammatory pain. METHODS: Baseline determinations of withdrawal from thermal and mechanical stimulation were made in Sprague-Dawley rats ( approximately 300-350 x g). Following determination of baseline, one hindpaw in each group was injected with 0.1 mL of CFA. The contralateral paw received saline. Thermal and mechanical stimulation was repeated on the third day post-injection. Vehicle (0.9% isotonic saline; pH 3.0) or ranolazine was then administered in randomized and blinded doses either by intraperitoneal (ip) injection (0, 10, 20, and 50 mg/kg) or by oral gavage (po; 0, 20, 50, 100, and 200 mg/kg). Animals were again tested 30 minutes (ip) and 1 hour (po) after drug administration. RESULTS: Ranolazine produced dose-dependant analgesia on mechanical allodynia induced by CFA injection, but had no effect on thermal hyperalgesia. CONCLUSIONS: Ranolazine's potential as a new option for managing both angina and chronic inflammatory pain warrants further study.

Up-regulation of AGS3 during morphine withdrawal promotes cAMP superactivation via adenylyl cyclase 5 and 7 in rat nucleus accumbens/striatal neurons.

Effective medical treatment of opiate addiction is limited by a high relapse rate in abstinent addicts. Opiate withdrawal causes cAMP superactivation, but the underlying molecular mechanisms are not clear. Recent evidence implicates an activator of G-protein signaling 3 (AGS3) in opiate addiction. We found previously that during a 10-min activation of opioid receptors, AGS3 binds G alpha(i)-GDP to promote free G betagamma stimulation of adenylyl cyclase (AC) 2 and 4, and/or inactivate G alpha(i) inhibitory function, thereby transiently enhancing cAMP-dependent protein kinase A (PKA) activity. In contrast, we report here that in nucleus accumbens/striatal neurons, morphine withdrawal induces cAMP superactivation, which requires up-regulation of AGS3. cAMP increases as a function of withdrawal time, by approximately 20% at 10 min and 75% at 5 h. However, cAMP superactivation does not require G betagamma. Instead, adenosine A2A receptor activation of G alpha(s/olf) seems to initiate cAMP superactivation and promote AGS3 up-regulation. Elevated AGS3 binds to G alpha(i) to prevent its inhibition on AC activation. Moreover, withdrawal-induced increases in cAMP/PKA activate phospholipase C and epsilon protein kinase C to further stimulate AC5 and AC7, causing cAMP superactivation. Our findings identify a critical role for AC 5 and 7 and A2A receptors for up-regulation of AGS3 in morphine withdrawal-induced cAMP superactivation.

Suppression of heavy drinking and alcohol seeking by a selective ALDH-2 inhibitor.

BACKGROUND: Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking. METHODS: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm. RESULTS: CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses. CONCLUSION: Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.

Ranolazine attenuates behavioral signs of neuropathic pain.

Ranolazine modulates the cardiac voltage-gated sodium channel (NaV 1.5) and is approved by the FDA in the treatment of ischemic heart disease. Ranolazine also targets neuronal (NaV 1.7, 1.8) isoforms that are implicated in neuropathic pain. Therefore, we determined the analgesic efficacy of ranolazine in a preclinical animal model of neuropathic pain. Both intraperitoneal and oral administration of ranolazine dose-dependently inhibited the mechanical and cold allodynia associated with spared nerve injury, without producing ataxia or other behavioral side effects. These data warrant clinical investigation of the potential use of ranolazine in the treatment of neuropathic pain.

Changing the paradigm: omegaven for the treatment of liver failure in pediatric short bowel syndrome.

BACKGROUND: Parenteral omega-3 fatty acids, such as Omegaven, may benefit patients with pediatric short bowel syndrome (SBS) who develop parenteral nutrition-associated liver disease (PNALD). PATIENTS AND METHODS: Retrospective cohort describing the outcome of all 12 children with SBS and advanced PNALD who were treated with Omegaven (target omega-6 to omega-3 fatty acid ratio = 1:1 to 2:1). RESULTS: The median age was 7.5 (range 3.6-46) months, and median parenteral nutrition duration before starting Omegaven was 28.4 (range 15.3-55.3) weeks. Median initial serum conjugated bilirubin was 137 (range 54-203) micromol/L (8.06 [3.18-11.94] mg/dL). Of the 12 patients, 9 had complete and sustained resolution of hyperbilirubinemia within a median of 24 (range 7-37) weeks, and all are no longer being considered for liver transplantation. Improvements in markers of hepatic inflammation as well as nutritional status also were noted in these patients. Three patients received a liver-intestine transplant while taking Omegaven. There were no complications attributable to Omegaven. CONCLUSIONS: Omegaven is associated with restoration of liver function in patients with SBS and advanced liver disease. Parenteral omega-3 fatty acids, such as Omegaven, have the potential to fundamentally alter the paradigm of neonatal SBS from one of early death or transplantation from liver failure to a more chronic disease. More children with SBS should achieve full enteral tolerance and those who do not have the capacity for intestinal adaptation should be able to survive and receive an intestinal graft when older.

Extravasation of intravenous computed tomography scan contrast in blunt abdominal and pelvic trauma.

BACKGROUND: Intravenous contrast extravasation (CE) on computed tomography (CT) scan in blunt abdominal trauma is generally regarded as an indication for the need for invasive intervention (either angiography or laparotomy). More recently, improvements in CT scan technology have increased the sensitivity in detecting CE, and, thus, we postulate that not all patients with this finding require intervention. METHODS: This study is a retrospective review of all patients who underwent a CT scan for blunt abdominal trauma between January 1999 and September 2003. Patterns of injury, associated injuries, management, and outcomes were examined for patients with CE. RESULTS: Seventy of 1,435 patients (4.8%) demonstrated CE. Mean age was 44 years and mean Injury Severity Score was 39. The location of CE was intra-abdominal in 25, pelvis/retroperitoneum in 39, and both areas in 3 patients. Six patients received supportive treatment for nonsurvivable head injury and were excluded from further analysis. Overall, 30 (47%) patients underwent immediate intervention (angiography or laparotomy) and 34 (53%) were managed nonoperatively. Of those who had initial nonoperative management, overall seven (20.5%) underwent intervention, with the remainder being managed without intervention. The success for nonoperative management was greater for those with pelvic/retroperitoneal CE (4 of 7: 57%) than for intra-abdominal extravasation (23 of 27: 85%). CONCLUSION: Although evidence of CE may suggest significant vascular injury, our data suggest that not all patients require invasive intervention. Further studies are needed to better define criteria for nonoperative management in patients with CE identified on their initial CT scan.

Preventable pediatric trauma deaths in Ontario: a comparative population-based study.

INTRODUCTION: Previously, we demonstrated that 21% of pediatric (<16 years) trauma deaths in the Province of Ontario during the period 1985 to 1987 were potentially preventable. Since then many trauma system changes have occurred including field triage, designation of trauma centers, and improved injury prevention. This study aims to examine the current preventable trauma death rate in our system using identical methodology to our previous study. METHOD: The records of all children (<16 years) who died in Ontario from 2001 to 2003 after blunt or penetrating trauma were obtained from the Chief Coroner and compared with those in our previous report. In both series, we excluded cases where care was not sought and all deaths due to asphyxia. Deaths were considered unpreventable if the Injury Severity Score, based on Abbreviated Injury Scale 1985, was >59; or if there was a head injury that received an Abbreviated Injury Scale score of 5 with the exception of isolated extra-axial hematomas. RESULTS: Eleven preventable deaths were identified. The preventable death rate was 7%, a significant decline from the 21% previously identified (p < 0.001; relative risk reduction for preventable death, 68% [95% confidence interval, 42-83%]; number needed to treat, 7). CONCLUSION: There has been a threefold decline in the preventable death rate, which we believe is related to improvements in the trauma system. We estimated that, for every seven deaths from fatal injuries, system changes between the two study periods eliminated one preventable death.

Assessing Competence in Emergency Radiology Using an Online Simulator.

RATIONALE AND OBJECTIVES: Traditional assessments in radiology residency focus on the Medical Expert CanMEDS role and typically rely upon a single or limited static images. We designed an Emergency Radiology Simulator that aimed to assess the breadth of competencies required across Medical and NonMedical Expert domains. MATERIAL AND METHODS: An online simulator with typical emergency cases was administered in October 2015 to Post Graduate Year (PGY) 2-5 residents in Radiology. Residents provided preliminary reports, which were graded for style and content. The simulation also included prioritization, protocoling, counseling, and handover exercises geared to assess NonMedical Expert roles. RESULTS: Fourty eight residents participated in the simulation. Level of resident was 11 PGY-2, 17 PGY-3, 13 PGY-4, and 7 PGY-5. There was a significant difference in resident performance between PGY-2 residents and those more senior in terms of the Medical Expert role (findings, diagnosis, recommendations, and clinical relevance of reports). Differences in performance between PGY levels were not seen in the NonMedical Expert roles (prioritization, protocoling, counseling, and handover). CONCLUSION: Simulation provides an opportunity to assess radiology resident performance across multiple domains. PGY-2 residents performed worse on the Medical Expert domains, although performance did not significantly vary between the other years. This may suggest that competence in Emergency Radiology is achieved early in residency, possibly related to the importance placed on developing skills related to on-call performance during the PGY-2 year. The simulator should be extended to other areas of Radiology, in order to assess the ability to discriminate performance in other subspecialties.

Dopamine and ethanol cause translocation of epsilonPKC associated with epsilonRACK: cross-talk between cAMP-dependent protein kinase A and protein kinase C signaling pathways.

We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.

The type 1 equilibrative nucleoside transporter regulates ethanol intoxication and preference.

Adenosine is an important mediator of ethanol intoxication. In vitro, ethanol stimulates adenosine signaling by inhibiting the type 1 equilibrative nucleoside transporter (ENT1), whereas chronic ethanol exposure downregulates ENT1. It is not known, however, whether ENT1 is important for ethanol intoxication or consumption in vivo. Here we report that ENT1-null mice show reduced hypnotic and ataxic responses to ethanol and greater consumption of alcohol as compared with their wild-type littermates. These features are associated with a decrease in adenosine tone, as measured indirectly as a reduction in A(1) receptor-mediated inhibition of glutamate excitatory postsynaptic currents (EPSCs) in the nucleus accumbens, leading to increased phosphorylation of CRE-binding protein (CREB) in the striatum. Treatment with an A(1) receptor agonist decreases EPSC amplitude and reduces ethanol consumption in ENT1-null mice. Our results indicate that ENT1 has a physiological role in ethanol-mediated behaviors and suggest that decreased A(1) adenosine receptor function promotes alcohol consumption.