Action Of Alcohol Research Articles

Prenatal Alcohol Exposure and Cortical Angiogenesis

Researchers at Normandy University, and Rouen and Brest Universities, France studied the effects of prenatal alcohol exposure on the cortical microvascular and the action of alcohol, glutamate, and vascular endothelial growth factor A (VEGF) on activity, plasticity, and survival of microvessels in mice.

Ethanol Reduces Neuronal Excitability of Lateral Orbitofrontal Cortex Neurons Via a Glycine Receptor Dependent Mechanism

Trauma-induced damage to the orbitofrontal cortex (OFC) often results in behavioral inflexibility and impaired judgment. Human alcoholics exhibit similar cognitive deficits suggesting that OFC neurons are susceptible to alcohol-induced dysfunction. A previous study from this laboratory examined OFC mediated cognitive behaviors in mice and showed that behavioral flexibility during a reversal learning discrimination task was reduced in alcohol-dependent mice. Despite these intriguing findings, the actions of alcohol on OFC neuron function are unknown. To address this issue, slices containing the lateral OFC (lOFC) were prepared from adult C57BL/6J mice and whole-cell patch clamp electrophysiology was used to characterize the effects of ethanol (EtOH) on neuronal function. EtOH (66 mM) had no effect on AMPA-mediated EPSCs but decreased those mediated by NMDA receptors. EtOH (11-66 mM) also decreased current-evoked spike firing and this was accompanied by a decrease in input resistance and a modest hyperpolarization. EtOH inhibition of spike firing was prevented by the GABAA antagonist picrotoxin, but EtOH had no effect on evoked or spontaneous GABA IPSCs. EtOH increased the holding current of voltage-clamped neurons and this action was blocked by picrotoxin but not the more selective GABAA antagonist biccuculine. The glycine receptor antagonist strychnine also prevented EtOH's effect on holding current and spike firing, and western blotting revealed the presence of glycine receptors in lOFC. Overall, these results suggest that acutely, EtOH may reduce lOFC function via a glycine receptor dependent process and this may trigger neuroadaptive mechanisms that contribute to the impairment of OFC-dependent behaviors in alcohol-dependent subjects.

Strong correlation between diet and development of colorectal cancer

Multiple factors have been described among the causes of non-hereditary colorectal cancer. In Western countries, the most common risk factors include upper-middle socioeconomic status and dietary regimens rich in proteins and animal fats. High consumption of red meats, smoked foods, cold cuts, or canned foods is believed to contribute to carcinogenesis as they directly affect epithlial turnover and cause metabolism of biliary acids. Dietary fibers have protective effects in that they capture the fats and biliary acids, thereby inhibiting their activity. Tobacco smoking acts both locally and systemically on the colorectal mucosa through the production of carcinogenic agents. Finally, the action of alcohol, in association with nicotine addiction, also increases the risk of developing colorectal tumors. Knowledge of dietary and environmental factors is of paramount importance in implementing preventive strategies for colorectal cancer.

Elucidating the biological basis for the reinforcing actions of alcohol in the mesolimbic dopamine system: the role of active metabolites of alcohol

The development of successful pharmacotherapeutics for the treatment of alcoholism is predicated upon understanding the biological action of alcohol. A limitation of the alcohol research field has been examining the effects of alcohol only and ignoring the multiple biological active metabolites of alcohol. The concept that alcohol is a “pro-drug” is not new. Alcohol is readily metabolized to acetaldehyde within the brain. Acetaldehyde is a highly reactive compound that forms a number of condensation products, including salsolinol and iso-salsolinol (acetaldehyde and dopamine). Recent experiments have established that numerous metabolites of alcohol have direct CNS action, and could, in part or whole, mediate the reinforcing actions of alcohol within the mesolimbic dopamine system. The mesolimbic dopamine system originates in the ventral tegmental area (VTA) and projects to forebrain regions that include the nucleus accumbens (Acb) and the medial prefrontal cortex (mPFC) and is thought to be the neurocircuitry governing the rewarding properties of drugs of abuse. Within this neurocircuitry there is convincing evidence that; (1) biologically active metabolites of alcohol can directly or indirectly increase the activity of VTA dopamine neurons, (2) alcohol and alcohol metabolites are reinforcing within the mesolimbic dopamine system, (3) inhibiting the alcohol metabolic pathway inhibits the biological consequences of alcohol exposure, (4) alcohol consumption can be reduced by inhibiting/attenuating the alcohol metabolic pathway in the mesolimbic dopamine system, (5) alcohol metabolites can alter neurochemical levels within the mesolimbic dopamine system, and (6) alcohol interacts with alcohol metabolites to enhance the actions of both compounds. The data indicate that there is a positive relationship between alcohol and alcohol metabolites in regulating the biological consequences of consuming alcohol and the potential of alcohol use escalating to alcoholism.

Ethanol-derived acetaldehyde: pleasure and pain of alcohol mechanism of action

Acetaldehyde (ACD), the first metabolite of ethanol (EtOH), has been implicated in several actions of alcohol, including its reinforcing effects. Previously considered an aversive compound, ACD was useful in alcoholic’s pharmacological treatment aimed at discouraging alcohol drinking. However, it has recently been shown that EtOH-derived ACD is necessary for EtOH-induced place preference and self-administration, thereby suggesting a possible involvement of ACD in EtOH motivational properties. In addition, EtOH-stimulating properties on DA neurons are prevented by pharmacological blockade of local catalase H2O2 system, the main metabolic step for biotransformation of EtOH into ACD within the central nervous system. It was further shown that pretreatment with thiol compounds, like L-Cysteine or D-Penicillamine, reduced EtOH and ACD-induced motivational effects, in fact preventing self-administration of both EtOH and ACD, thus suggesting a possible role for ACD as a biomarker useful in evaluating potential innovative treatments of alcohol abuse. These findings suggest a key role of ACD in the EtOH reinforcing effects. In the present paper we review the role of EtOH-derived ACD in the reinforcing effects of EtOH and the possibility that ACD may serve as a therapeutically targetable biomarker in the search for novel treatments in alcohol abuse and alcoholism.

Primate cerebellar granule cells exhibit a tonic GABAAR conductance that is not affected by alcohol: a possible cellular substrate of the low level of response phenotype

In many rodent brain regions, alcohol increases vesicular release of GABA, resulting in an increase in the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) and the magnitude of tonic GABAA receptor (GABAAR) currents. A neglected issue in translating the rodent literature to humans is the possibility that phylogenetic differences alter the actions of alcohol. To address this issue we made voltage-clamp recordings from granule cells (GCs) in cerebellar slices from the non-human primate (NHP), Macaca fascicularis. We found that similar to Sprague Dawley rats (SDRs), NHP GCs exhibit a tonic conductance generated by α6δ subunit containing GABAARs, as evidenced by its blockade by the broad spectrum GABAAR antagonist, GABAzine (10 μM), inhibition by α6 selective antagonist, furosemide (100 μM), and enhancement by THDOC (10–20 nM) and THIP (500 nM). In contrast to SDR GCs, in most NHP GCs (~60%), application of EtOH (25–105 mM) did not increase sIPSC frequency or the tonic GABAAR current. In a minority of cells (~40%), EtOH did increase sIPSC frequency and the tonic current. The relative lack of response to EtOH was associated with reduced expression of neuronal nitric oxide synthase (nNOS), which we recently reported mediates EtOH-induced enhancement of vesicular GABA release in rats. The EtOH-induced increase in tonic GABAAR current was significantly smaller in NHPs than in SDRs, presumably due to less GABA release, because there were no obvious differences in the density of GABAARs or GABA transporters between SDR and NHP GCs. Thus, EtOH does not directly modulate α6δ subunit GABAARs in NHPs. Instead, EtOH enhanced GABAergic transmission is mediated by enhanced GABA release. Further, SDR GC responses to alcohol are only representative of a subpopulation of NHP GCs. This suggests that the impact of EtOH on NHP cerebellar physiology will be reduced compared to SDRs, and will likely have different computational and behavioral consequences.

Alcohol and NMDA receptor: current research and future direction

The brain is one of the major targets of alcohol actions. Most of the excitatory synaptic transmission in the central nervous system is mediated by N-methyl-D-aspartate (NMDA) receptors. However, one of the most devastating effects of alcohol leads to brain shrinkage, loss of nerve cells at specific regions through a mechanism involving excitotoxicity, oxidative stress. Earlier studies have indicated that chronic exposure to ethanol both in vivo and in vitro, increases NR1 and NR2B gene expression and their polypeptide levels. The effect of alcohol and molecular changes on the regulatory process, which modulates NMDAR functions including factors altering transcription, translation, post-translational modifications, and protein expression, as well as those influencing their interactions with different regulatory proteins (downstream effectors) are incessantly increasing at the cellular level. Further, I discuss the various genetically altered mice approaches that have been used to study NMDA receptor subunits and their functional implication. In a recent countable review, epigenetic dimension (i.e., histone modification-induced chromatin remodeling and DNA methylation, in the process of alcohol related neuroadaptation) is one of the key molecular mechanisms in alcohol mediated NMDAR alteration. Here, I provide a recount on what has already been achieved, current trends and how the future research/studies of the NMDA receptor might lead to even greater engagement with many possible new insights into the neurobiology and treatment of alcoholism.

Activating Ion Determines Differential Ethanol-Sensitivity of Slo Family Channels

Ethanol levels reached in circulation during moderate-heavy alcohol intoxication (50 mM) modify BK channel steady-state activity (NPo) eventually altering physiology and behavior. In general, ethanol decreases NPo of vascular smooth muscle BK channels while increasing NPo of neuronal BK channels. Channel subunit composition, posttranslational modification and channel lipid microenvironment all play a role in alcohol final effect (Brodie et al., 2007). However, ethanol action on BK NPo solely requires the channel-forming subunit (slo1) and is function of activating ion (i.e., Ca2+i) (Liu et al., 2008; Bukiya et al., 2009). Whether activating ion-dependence of alcohol action is unique to slo1 or, rather, extends to the other members of the slo family remains unknown. Slo2.1 (Slick), Slo2.2 (Slack), and Slo3 gene products render ion channel proteins that display a phenotype common to slo1: high conductance for K+ and dual regulation of gating by transmembrane voltage and intracellular ion-recognition, with slo2 and slo3 being activated by Na+i and OH−i, respectively (Salkoff et al., 2006). Here, after expression in Xenopus oocytes, we probed cbv1, slick/slack and slo3 channels with 50 mM ethanol over a wide range of activating ion concentration in inside-out patches. Thus, we constructed conductance-voltage plots from macroscopic current or measured NPo from single channel recordings. Ethanol-induced potentiation of cbv1 currents turned into inhibition as Ca2+i increased, with a crossover at ∼20 μM Ca2+i. In contrast, slo2 and slo3 channels remained resistant to 50 mM ethanol even when probed at a wide concentration range of activating ion: 10-140 mM Na+i (slo2) and 0.01-10 μM OH−i (slo3). Data underscore that the ethanol-sensitivity and its ligand-dependence found in slo1 channels are not extensive to other members of the slo channel family, suggesting selective interaction(s) between Ca2+-sensing and ethanol-sensing mechanisms. Support: R37AA011560 (AMD)

A Hard Road: Driving Local Action against Alcohol Related Problems in a Rural Town

Context is important in developing strategies to address alcohol related violence. Knowledge of local conditions is critical to action in rural areas. The aim of this study was to gather information about context specific alcohol related problems experienced by frontline workers in a regional centre to inform the local alcohol action plan. Frontline workers were invited to participate in one of five focus group discussions that investigated problems experienced as a result of other people’s alcohol use. Alcohol related problems were more frequently associated with time periods than any single group in the community. Social media was used to incite arguments between groups in different venues during the lock-out periods. The focus groups identified that the location of licensed premises and a taxi rank; and previous relationships between protagonists were the key contextual factors causing alcohol related problems. A second taxi rank was identified as a useful local management strategy. Supply reduction was suggested as a key factor in long term solutions to alcohol related problems in rural towns. The local liquor accord did not want to reduce supply of alcohol by closing late night venues earlier. Local action to reduce alcohol related problems will be limited to pragmatic solutions because supply reduction is unacceptable to those in the business of selling alcohol.

Prenatal Alcohol Exposure Affects Vasculature Development in the Neonatal Brain

In humans, antenatal alcohol exposure elicits various developmental disorders, in particular in the brain. Numerous studies focus on the deleterious effects of alcohol on neural cells. Although recent studies suggest that alcohol can affect angiogenesis in adults, the impact of prenatal alcohol exposure on brain microvasculature remains poorly understood. We used a mouse model to investigate effects of prenatal alcohol exposure on the cortical microvascular network in vivo and ex vivo and the action of alcohol, glutamate, and vascular endothelial growth factor A (VEGF) on activity, plasticity, and survival of microvessels. We used quantitative reverse transcriptase polymerase chain reaction, Western blot, immunohistochemistry, calcimetry, and videomicroscopy. We characterized the effect of prenatal alcohol exposure on the cortical microvascular network in human controls and fetal alcohol syndrome (FAS)/partial FAS (pFAS) patients at different developmental stages. In mice, prenatal alcohol exposure induced a reduction of cortical vascular density, loss of the radial orientation of microvessels, and altered expression of VEGF receptors. Time-lapse experiments performed on brain slices revealed that ethanol inhibited glutamate-induced calcium mobilization in endothelial cells, affected plasticity, and promoted death of microvessels. These effects were prevented by VEGF. In humans, we evidenced a stage-dependent alteration of the vascular network in the cortices of fetuses with pFAS/FAS. Whereas no modification was observed from gestational week 20 (WG20) to WG22, the radial organization of cortical microvessels was clearly altered in pFAS/FAS patients from WG30 to WG38. Prenatal alcohol exposure affects cortical angiogenesis both in mice and in pFAS/FAS patients, suggesting that vascular defects contribute to alcohol-induced brain abnormalities.

The Small G Protein H-Ras in the Mesolimbic System Is a Molecular Gateway to Alcohol-Seeking and Excessive Drinking Behaviors

Uncontrolled consumption of alcohol is a hallmark of alcohol abuse disorders; however, the central molecular mechanisms underlying excessive alcohol consumption are still unclear. Here, we report that the GTP binding protein, H-Ras in the nucleus accumbens (NAc) plays a key role in neuroadaptations that underlie excessive alcohol-drinking behaviors. Specifically, acute (15 min) systemic administration of alcohol (2.5 g/kg) leads to the activation of H-Ras in the NAc of mice, which is observed even 24 h later. Similarly, rat operant self-administration of alcohol (20%) also results in the activation of H-Ras in the NAc. Using the same procedures, we provide evidence suggesting that the exchange factor GRF1 is upstream of H-Ras activation by alcohol. Importantly, we show that infection of mice NAc with lentivirus expressing a short hairpin RNA that targets the H-Ras gene produces a significant reduction of voluntary consumption of 20% alcohol. In contrast, knockdown of H-Ras in the NAc of mice did not alter water, quinine, and saccharin intake. Furthermore, using two-bottle choice and operant self-administration procedures, we show that inhibiting H-Ras activity by intra-NAc infusion of the farnesyltransferase inhibitor, FTI-276, produced a robust decrease of rats' alcohol drinking; however, sucrose consumption was unaltered. Finally, intra-NAc infusion of FTI-276 also resulted in an attenuation of seeking for alcohol. Together, these results position H-Ras as a central molecular mediator of alcohol's actions within the mesolimbic system and put forward the potential value of the enzyme as a novel target to treat alcohol use disorders.

Independent contributions of alcohol and stress axis hormones to painful peripheral neuropathy

Painful small-fiber peripheral neuropathy is a debilitating complication of chronic alcohol abuse. Evidence from previous studies suggests that neuroendocrine mechanisms, in combination with other, as yet unidentified actions of alcohol, are required to produce this neuropathic pain syndrome. In addition to neurotoxic effects of alcohol, in the setting of alcohol abuse neuroendocrine stress axes release glucocorticoids and catecholamines. Since receptors for these stress hormones are located on nociceptors, at which they can act to cause neuronal dysfunction, we tested the hypothesis that alcohol and stress hormones act on the nociceptor, independently, to produce neuropathic pain. We used a rat model, which allows the distinction of the effects of alcohol from those produced by neuroendocrine stress axis mediators. We now demonstrate that topical application of alcohol and exposure to unpredictable sound stress, each alone, has no effect on the nociceptive threshold. However, when animals that had previous exposure to alcohol were subsequently exposed to stress, they rapidly developed mechanical hyperalgesia. Conversely, sound stress followed by topical alcohol exposure also produced mechanical hyperalgesia. The contribution of stress hormones was prevented by spinal intrathecal administration of oligodeoxynucleotides antisense to β2-adrenergic or glucocorticoid receptor mRNA, which attenuates receptor level in nociceptors, as well as by adrenal medullectomy. These experiments establish an independent role of alcohol and stress hormones on the primary afferent nociceptor in the induction of painful peripheral neuropathy.

Introducing nurse prescribing in a substance misuse treatment service

In this article, a project commissioned by a drug and alcohol action team in the south west of England is described. The project brief is to implement non-medical prescribing (NMP) within a substance misuse treatment service in order to increase flexibility of resources for delivery of care. All nurses working within the substance misuse service who are paid at agenda for change band 6 are involved in delivery ( NHS Careers, 2012 ). It is the intention that all will take on a prescribing role, which will introduce more than 10 non-medical prescribers. The project is linked to a strategically led service redesign; one of the aims of which is to improve access to the substance misuse service in isolated rural areas ( Cornwall and Isles of Scilly Primary Care Trust and Drug and Alcohol Action Team, 2010 ). This article outlines the background for the change; the strategies used to implement the project, particularly those used to motivate and involve participants including users of services; and the agreed deliverables. It concludes with a critical evaluation of the project progress to date, together with an outline of how the new arrangements will be sustained and measured, and lessons learned for future similar projects. The project, which started life as a NMP project, has been developed in to an overarching ‘prescribing project’. An additional benefit of the project has been increased engagement and acceptance of change.

Neuronal Nitric Oxide Synthase Mediates the Effect of Ethanol on IgA

We showed previously that the acute effect of ethanol on intestinal immunoglobulin A (IgA) expression might be mediated by endogenous nitric oxide (NO). To extend these findings, this study was designed to investigate a possible role of neuronal NO synthase (nNOS) in the observed phenomenon, using 7-nitroindazole (7-NI), a selective inhibitor of its activity. Adult male Wistar rats were treated with: (a) ethanol (4g/kg, intraperitoneally, i.p.), (b) 7-NI (25mg/kg, i.p.) followed by ethanol (4g/kg, i.p.) 30min later and (c) 7-NI (25mg/kg, i.p.) followed by saline 30min later. Untreated rats were used as controls. The concentrations of serum and intestinal IgA were measured by enzyme-linked immunosorbent assay, while the expression of nNOS was determined using western blot and immunohistochemistry. Acute ethanol treatment significantly increased the concentration of IgA in the ileal extracts, whereas it decreased its serum level. Inhibition of nNOS activity by 7-NI abolished this action of alcohol on IgA. Additionally, western blot analysis revealed that the acute alcohol administration induced an increase in the expression of intestinal nNOS. Furthermore, nNOS-immunoreactive cells, observed within the lamina propria of small intestine, were numerous in ethanol-treated rats. Taken together, these results extended our previous findings suggesting that nNOS mediates the acute effect of ethanol on IgA and supported an immunomodulatory role of this enzyme isoform.

Screening and brief intervention delivered simultaneously in multiple settings: it is cost-effective, but can it influence community-level outcomes?

A 20-community randomized controlled trial, the Alcohol Action in Rural Communities (AARC) project, provided the opportunity to examine the cost-effectiveness of screening and brief intervention (SBI) delivered simultaneously in general practice (GP), pharmacy, and emergency department (ED) settings and the community level impact of the SBI on problem drinking. For the GP- and pharmacy-delivered SBI, decision models and scenario analysis assessed outcomes and costs in the 10 experimental communities of the trial. For the ED-delivered SBI, a randomized controlled trial design was used to examine the cost-effectiveness of mailed personalized feedback. For both the GP- and pharmacy-delivered SBI, the most cost-effective outcome was to increase screening alone: GPs and pharmacies screening all patients achieved an incremental cost-effectiveness ratio (ICER) of AUD $197 and AUD $29, respectively, per risky drinker who reduced drinking. The ED-based SBI resulted in a reduction of 2.6 fewer drinks per week at an average cost of $5.55 per patient and an ICER of $2.13 per one standard drink reduction in average weekly consumption. In addition to cost-effectiveness, the AARC community approach provided the opportunity to analyze the effect of SBI on community level outcomes. Currently, 19% of risky drinkers in a community visit a GP and reduce their drinking, which would increase to 36% if all patients got SBI. Similarly, 23% of risky drinkers in a community visit a pharmacy and reduce their drinking, which would increase to 34% if they all got SBI. Although our results confirm SBI is cost-effective, the impact at the community level is unclear: if all GPs and pharmacists delivered SBI to all their risky drinking patients, only 34-36% would reduce their drinking. A trial that assessed the impact of SBI delivered in multiple settings simultaneously on community level indicators of alcohol harm would move the field toward demonstrating the cost benefit, as well as cost-effectiveness, of SBI.

A brief intervention targeting primary-care physician prescribing of pharmacotherapies for alcohol dependence: can it impact prescribing behavior and reduce hospital inpatient admissions?

Increasing the use of pharmacotherapies for alcohol dependence has the potential to improve patient outcomes and reduce health-care costs by reducing hospital admissions. This randomized controlled trial (RCT) evaluated the cost-effectiveness of tailored mailed feedback on general practitioner (GP) prescribing for alcohol dependence and alcohol-related hospital admissions. General practitioners (N = 115) in 10 communities randomized to the experimental arm of the Alcohol Action in Rural Communities (AARC) project received tailored mailed feedback on their prescribing of acamprosate and naltrexone. Segmented regression analysis examined the impact of the intervention relative to GPs’ prescribing and inpatient hospital admissions for alcohol dependence in those communities. Incremental cost-effectiveness ratios were estimated to compare costs per additional prescription written and costs per inpatient admission averted. Trend analysis showed GPs significantly increased their prescribing of acamprosate (s = 0.24; 95% confidence interval [CI], 0.13-0.35) and significantly decreased their prescribing of naltrexone (s = 0.12; 95% CI, 0.13-0.35). Rates of alcohol-related inpatient admissions for alcohol dependence decreased significantly in the experimental group compared with the control group (s = 0.98; 95% CI, 1.80-0.16). Similar to evidence showing SBI can improve patient outcomes, this study showed mailed tailored feedback to GPs achieved cost-effective increases in their prescribing of acamprosate, with a subsequent and plausibly causal reduction in inpatient hospital admissions for alcohol dependence. Demonstrating the capacity of brief intervention in primary-care settings to reduce demand for tertiary care services appears to be a promising direction for the SBI field. A large-scale RCT of the cost benefit of tailored feedback to GPs appears warranted.

Alcohol-induced alterations in maternal uterine endothelial proteome: A quantitative iTRAQ mass spectrometric approach

ObjectiveTo quantitate alcohol-induced alterations in the maternal uterine endothelial proteome utilizing iTRAQ-based mass spectrometry. Study designUterine artery endothelial cells from third trimester pregnant ewes were FAC sorted, validated and treated without or with binge-like alcohol. Lysates were trypsin digested, iTRAQ-labeled, and analyzed using nano LC MS/MS. ResultsAlcohol significantly upregulated 14 and downregulated 17 proteins (P<0.05) including those related to cell structure, transcription/translation regulation, histones, Ca2+/NO, and redox balance. Gene Ontology and ArrayTrack analyses revealed alterations to protein processing, binding, and nutrient metabolism pathways. Further, alcohol altered proteins previously correlated with fetal alcohol spectrum disorders (FASD) and those that regulate epigenetic, transcriptional, and translational processes. ConclusionsAlcohol differentially alters the proteome in the maternal uterine compartment at the level of the endothelium. iTRAQ mass spectrometry provides a robust high throughput platform to comprehend the multi-mechanistic actions of alcohol and develop appropriate biomarkers and ameliorative measures for FASD.

Is a cardioprotective action of alcohol a myth?

Numerous epidemiological studies have demonstrated that light to moderate alcohol consumption is associated with reduced risk of cardiovascular disease (CVD). The purpose of this review is to discuss the potential CV benefit of alcohol consumption with a particular focus on the findings of publications appearing within the past 2 years. Results of observational studies and meta-analyses are largely concordant in suggesting the possibility of a beneficial effect of alcohol on CVD via several mechanisms, including actions on lipid metabolism and hemostatic factors. There are, however, studies that do not find the classic U-shaped association between alcohol consumption and CVD. Recent data suggest that the findings of coronary protection by alcohol consumption may be partly due to misclassification and confounding factors. Drinking patterns appear to be important factors to take into account when interpreting the results of epidemiological studies. No data are currently available to directly support a causal relationship between alcohol intake and CVD. As well-conducted randomized studies assessing the causal role of alcohol in cardioprotection are not feasible, future epidemiological studies evaluating the relationship between alcohol and CVD should carefully choose the covariates in any multivariate analysis. It remains premature to promote alcohol consumption as a basis for CV protection.

Interactions among Positions in the Third and Fourth Membrane-associated Domains at the Intersubunit Interface of the N-Methyl-d-aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces.

Respiratory depression in rats induced by alcohol and barbiturate and rescue by ampakine CX717

Barbiturate use in conjunction with alcohol can result in severe respiratory depression and overdose deaths. The mechanisms underlying the additive/synergistic actions were unresolved. Current management of ethanol-barbiturate-induced apnea is limited to ventilatory and circulatory support coupled with drug elimination. Based on recent preclinical and clinical studies of opiate-induced respiratory depression, we hypothesized that ampakine compounds may provide a treatment for other types of drug-induced respiratory depression. The actions of alcohol, pentobarbital, bicuculline, and the ampakine CX717, alone and in combination, were measured via 1) ventral root recordings from newborn rat brain stem-spinal cord preparations and 2) plethysmographic recordings from unrestrained newborn and adult rats. We found that ethanol caused a modest suppression of respiratory drive in vitro (50 mM) and in vivo (2 g/kg ip). Pentobarbital induced an ∼50% reduction in respiratory frequency in vitro (50 μM) and in vivo (28 mg/kg for pups and 56 mg/kg for adult rats ip). However, severe life-threatening apnea was induced by the combination of the agents in vitro and in vivo via activation of GABA(A) receptors, which was exacerbated by hypoxic (8% O(2)) conditions. Administration of the ampakine CX717 alleviated a significant component of the respiratory depression in vitro (50-150 μM) and in vivo (30 mg/kg ip). Bicuculline also alleviated ethanol-/pentobarbital-induced respiratory depression but caused seizure activity, whereas CX717 did not. These data demonstrated that ethanol and pentobarbital together caused severe respiratory depression, including lethal apnea, via synergistic actions that blunt chemoreceptive responses to hypoxia and hypercapnia and suppress central respiratory rhythmogenesis. The ampakine CX717 markedly reduced the severity of respiratory depression.