Adrienne Gordon

βγ Dimers Mediate Synergy of Dopamine D2 and Adenosine A2 Receptor-Stimulated PKA Signaling and Regulate Ethanol Consumption

Dopamine release is activated by ethanol and addicting drugs, but molecular mechanisms linking dopaminergic signaling to neuronal responses and drinking behavior are poorly understood. We report that dopamine-D2 receptors induce PKA Calpha translocation and increase CRE-regulated gene expression. Ethanol also activates PKA signaling. Subthreshold concentrations of the D2 agonist NPA and ethanol, without effect alone, together cause synergistic PKA translocation and CRE-mediated gene transcription. D2 or adenosine A2 receptor blockade, pertussis toxin, Rp-cAMPS, or overexpression of dominant-negative peptides that sequester betagamma dimers prevent synergy. Importantly, overexpression of a betagamma inhibitor peptide in the nucleus accumbens strikingly reduces sustained alcohol consumption. We propose that synergy of D2 and A2 confers ethanol hypersensitivity and that betagamma dimers are required for voluntary drinking.

An evaluation of classification systems for stillbirth.

BackgroundAudit and classification of stillbirths is an essential part of clinical practice and a crucial step towards stillbirth prevention. Due to the limitations of the ICD system and lack of an international approach to an acceptable solution, numerous disparate classification systems have emerged. We assessed the performance of six contemporary systems to inform the development of an internationally accepted approach.MethodsWe evaluated the following systems: Amended Aberdeen, Extended Wigglesworth; PSANZ-PDC, ReCoDe, Tulip and CODAC. Nine teams from 7 countries applied the classification systems to cohorts of stillbirths from their regions using 857 stillbirth cases. The main outcome measures were: the ability to retain the important information about the death using the InfoKeep rating; the ease of use according to the Ease rating (both measures used a five-point scale with a score <2 considered unsatisfactory); inter-observer agreement and the proportion of unexplained stillbirths. A randomly selected subset of 100 stillbirths was used to assess inter-observer agreement.ResultsInfoKeep scores were significantly different across the classifications (p ≤ 0.01) due to low scores for Wigglesworth and Aberdeen. CODAC received the highest mean (SD) score of 3.40 (0.73) followed by PSANZ-PDC, ReCoDe and Tulip [2.77 (1.00), 2.36 (1.21), 1.92 (1.24) respectively]. Wigglesworth and Aberdeen resulted in a high proportion of unexplained stillbirths and CODAC and Tulip the lowest. While Ease scores were different (p ≤ 0.01), all systems received satisfactory scores; CODAC received the highest score. Aberdeen and Wigglesworth showed poor agreement with kappas of 0.35 and 0.25 respectively. Tulip performed best with a kappa of 0.74. The remainder had good to fair agreement.ConclusionThe Extended Wigglesworth and Amended Aberdeen systems cannot be recommended for classification of stillbirths. Overall, CODAC performed best with PSANZ-PDC and ReCoDe performing well. Tulip was shown to have the best agreement and a low proportion of unexplained stillbirths. The virtues of these systems need to be considered in the development of an international solution to classification of stillbirths. Further studies are required on the performance of classification systems in the context of developing countries. Suboptimal agreement highlights the importance of instituting measures to ensure consistency for any classification system.

Causes of death and associated conditions (Codac) – a utilitarian approach to the classification of perinatal deaths

A carefully classified dataset of perinatal mortality will retain the most significant information on the causes of death. Such information is needed for health care policy development, surveillance and international comparisons, clinical services and research. For comparability purposes, we propose a classification system that could serve all these needs, and be applicable in both developing and developed countries. It is developed to adhere to basic concepts of underlying cause in the International Classification of Diseases (ICD), although gaps in ICD prevent classification of perinatal deaths solely on existing ICD codes.We tested the Causes of Death and Associated Conditions (Codac) classification for perinatal deaths in seven populations, including two developing country settings. We identified areas of potential improvements in the ability to retain existing information, ease of use and inter-rater agreement. After revisions to address these issues we propose Version II of Codac with detailed coding instructions.The ten main categories of Codac consist of three key contributors to global perinatal mortality (intrapartum events, infections and congenital anomalies), two crucial aspects of perinatal mortality (unknown causes of death and termination of pregnancy), a clear distinction of conditions relevant only to the neonatal period and the remaining conditions are arranged in the four anatomical compartments (fetal, cord, placental and maternal).For more detail there are 94 subcategories, further specified in 577 categories in the full version. Codac is designed to accommodate both the main cause of death as well as two associated conditions. We suggest reporting not only the main cause of death, but also the associated relevant conditions so that scenarios of combined conditions and events are captured.The appropriately applied Codac system promises to better manage information on causes of perinatal deaths, the conditions associated with them, and the most common clinical scenarios for future study and comparisons.

Addicting drugs utilize a synergistic molecular mechanism in common requiring adenosine and Gi-βγ dimers

The mesolimbic dopamine system and cAMP-dependent/protein kinase A (PKA) pathways are strongly implicated in addictive behaviors. Here we determine the role of dopamine D2 receptors (D2) in PKA signaling responses to δ-opioid (DOR) and cannabinoid (CB1) receptors. We find in NG108-15/D2 cells and in cultured primary neurons that a brief exposure to saturating concentrations of DOR and CB1 agonists increases cAMP, promotes PKA Cα translocation and increases cAMP-dependent gene expression. Activation of PKA signaling is mediated by Gi-βγ dimers. Importantly, subthreshold concentrations of DOR or CB1 agonists with D2 agonists, which are without effect when added separately, together activate cAMP/PKA signaling synergistically. There is also synergy between DOR or CB1 with ethanol, another addicting agent. In all instances, synergy requires adenosine activation of adenosine A2 receptors and is mediated by βγ dimers. Synergy by this molecular mechanism appears to confer hypersensitivity to opioids and cannabinoids while simultaneously increasing the sensitivity of D2 signaling when receptors are expressed on the same cells. This mechanism may account, in part, for drug-induced activation of medium spiny neurons in the nucleus accumbens.

Ethanol and the γ-Aminobutyric Acid-Benzodiazepine Receptor Complex

Abstract: Ethanol appears to enhance γ‐aminobutyric acid (GABA)‐mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA‐benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 mM failed to alter binding of [3H]flunitrazepam (FNZ), [3H]Ro 15‐1788, or [3H]methyl‐γ‐carboline‐3‐carboxylate (MBCC) to benzodiazepine receptors, or of [3H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4°C and 37°C revealed no significant effects of 100 mM ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [3H]FNZ and/or [3H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA‐benzodiazepine receptor complex.

The Role of Protein Kinase C in Cellular Tolerance to Ethanol

We have shown that ethanol inhibits uptake of adenosine by a specific nucleoside transporter in NG108-15 neuroblastoma × glioma cells and that cAMP-dependent protein kinase (PKA) activity is required for this inhibition. After chronic exposure to ethanol, adenosine uptake is no longer inhibited on rechallenge with ethanol, i.e. transport has become tolerant to ethanol. Here we show that protein kinase C (PKC) contributes to ethanol-induced tolerance of adenosine transport. Activation of PKC by phorbol esters in control cells results in an ethanol-tolerant phenotype, similar to that produced by chronic ethanol exposure. In addition, chronic exposure to ethanol increases the amounts of α, δ, and ε PKC. However, reducing PKC activity by inhibition with chelerythrine during chronic exposure to ethanol or down-regulation by phorbol esters prevents the development of ethanol-induced tolerance of adenosine transport. By contrast, the inhibition of PKA activity produces tolerance to ethanol inhibition of adenosine uptake. When protein phosphatase inhibitors are present, inhibiting PKA activity has no effect on ethanol sensitivity of adenosine uptake, suggesting a role for protein phosphatases in the regulation of ethanol sensitivity of uptake. Taken together, our results suggest that PKA and PKC have opposing effects on the ethanol sensitivity of adenosine transport; PKA activity is required for ethanol sensitivity, and PKC activation produces tolerance. Based on these data, we propose that chronic ethanol exposure increases PKC activity, leading to the activation of a protein phosphatase (1 or 2A). This phosphatase then dephosphorylates a PKA-phosphorylated site, which is required for ethanol to inhibit adenosine uptake. Therefore, the sensitivity of adenosine transport to ethanol appears to be maintained by a balance of PKA and protein phosphatase activities, and PKC may regulate phosphatase activity.

Late onset neonatal gram-negative bacillary infection in Australia and New Zealand: 1992-2002

Background: Late onset neonatal Gram-negative bacillary infection is a significant cause of morbidity and mortality. Objective: To determine the incidence and mortality from late onset Gram-negative bacillary infections in neonatal units. Methods: An 11-year longitudinal prospective surveillance study. Clinicians in 20 neonatal units in Australia and New Zealand collected data. Late onset sepsis was defined as clinical signs of infection starting more than 48 hours after birth, with laboratory evidence supporting sepsis and pure growth of a pathogen from blood and/or cerebrospinal fluid. Results: Overall, 702 of 3113 (22.5%) episodes of late onset sepsis in 681 infants were from Gram-negative bacilli. Overall mortality was 20.8% (142 of 681 infants) and significantly related to maturity, birth weight and infecting organism. Mortality was 25% for infants <30 weeks compared with 11.5% for infants ≥30 weeks gestation (P < 0.0001) and 24.2% for infants with birth weights <1500 g versus 12.7% if ≥1500 g (P < 0.0001). Infection by Pseudomonas aeruginosa was associated with 52.3% mortality (46 of 88 infants), significantly higher than the 13.7% to 23.8% fatality from other Gram-negative bacilli (P < 0.0001). During the surveillance, the late onset Gram-negative bacillary infection rate remained stable at 1.14 per 1000 live births (range 0.87–1.5). Similarly, mortality was unchanged, being 0.25 per 1000 live births (range 0.12–0.43). Conclusions: Gram-negative bacilli remain important causes of late onset neonatal sepsis, especially among very low birth weight infants, and result in a high mortality, particularly with P. aeruginosa infections.

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 ϵ protein kinase C (ϵPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of ϵPKC and the relationship to PKA activation. In the present study, we used a new ϵPKC antibody, 14E6, that selectively recognized active ϵPKC when not bound to its anchoring protein ϵRACK (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 ϵPKC and induced translocation of both ϵPKC and its anchoring protein, ϵRACK to a new cytosolic site. The selective ϵPKC agonist, pseudo-ϵRACK, activated ϵPKC but did not cause translocation of the ϵPKC/ϵRACK complex to the cytosol. These data suggest a step-wise activation and translocation of ϵPKC after NPA or ethanol treatment, where ϵPKC first translocates and binds to its RACK and subsequently the ϵPKC/ϵRACK 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 ϵPKC 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 ϵPKC 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.

Sleep position, fetal growth restriction, and late-pregnancy stillbirth: the Sydney stillbirth study.

OBJECTIVE: To identify potentially modifiable risk factors for late-pregnancy stillbirth. METHODS: This was a population-based matched case–control study of pregnant women at 32 weeks of gestation or greater booked into tertiary maternity hospitals in metropolitan Sydney between January 2006 and December 2011. The case group consisted of women with singleton pregnancies with antepartum fetal death in utero. Women in the control group were matched for booking hospital and expected delivery date with women in the case group. Data collection was performed using a semistructured interview and included validated questionnaires for specific risk factors. Adjusted odds ratios (ORs) were calculated for a priori-specified risk factors using conditional logistic regression. RESULTS: There were 103 women in the case group and 192 women in the control group. Mean gestation was 36 weeks. Supine sleeping was reported by 10 of 103 (9.7%) of women who experienced late-pregnancy stillbirth and by 4 of 192 (2.1%) of women in the control group (adjusted OR 6.26, 95% confidence interval [CI] 1.2–34). Women who experienced stillbirth were more likely to: have been followed during pregnancy for suspected fetal growth restriction, 11.7% compared with 1.6% (adjusted OR 5.5, 95% CI 1.36–22.5); not be in paid work, 25.2% compared with 9.4% (adjusted OR 2.9, 95% CI 1.1–7.6); and to have not received further education beyond high school, 41.7% compared with 25.5% (adjusted OR 1.9, 95% CI 1.1–3.5). None of the deaths to women who reported supine sleeping were classified as unexplained. CONCLUSION: This study suggests that supine sleep position may be an additional risk for late-pregnancy stillbirth in an already compromised fetus. The clinical management of suspected fetal growth restriction should be investigated further as a means of reducing late stillbirth. LEVEL OF EVIDENCE: II

Late-onset infection and the role of antibiotic prescribing policies

Purpose of review Late-onset infection is a significant cause of morbidity and mortality in low-birth-weight and premature infants. Empirical antibiotic treatment is used as infants can deteriorate rapidly without treatment. Current data on the epidemiology of late-onset infection, the types of antibiotics used, duration of antibiotic use, and antibiotic prescribing policies are reviewed. Recent findings Epidemiological data on late-onset sepsis is dominated by information concerning developed countries; large prospective data collections have been set up in many such countries. Recent data indicate that late-onset sepsis occurs in one-fifth of very-low-birth-weight infants. There are increasing concerns regarding antibiotic resistance. Antibiotic regimens that do not include third-generation cephalosporins produce less resistance. Strategies of antibiotic rotation have not been documented as producing a marked effect on the development of resistant micro-organisms, but there is a lack of randomized trials. Recommendations for preventing the spread of vancomycin-resistant enterococci, produced by the Hospital Infection Control Practices Advisory Committee, have been shown to be effective in a number of situations. Recent reports have documented the success of multidisciplinary, systems-orientated approaches for reducing neonatal nosocomial infection. Summary Antibiotic prescribing policies have an important role to play in the treatment of late-onset neonatal infection. There is enough evidence to state that narrow-spectrum antibiotics should be used wherever possible and that potent broad-spectrum antibiotics should be kept in reserve. Ongoing prospective surveillance of infection rates, micro-organisms, resistance and antibiotic use is essential.