Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Biomimetic reconstruction of the hematopoietic stem cell niche for in vitro amplification of human hematopoietic stem cells.

Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.

Time of out-of-hospital cardiac arrest is not associated with outcome in a metropolitan area: A multicenter cohort study.

AIM: Whether time of day influences survival after out-of-hospital cardiac arrest (OHCA) remains controversial. We compared outcomes after OHCA between day and night and explored whether characteristics of pre-hospital advanced life support (ALS)-quality varied by time of day. METHODS: We conducted a prospective cohort study of individuals that suffered a non-traumatic OHCA in the city of Vienna between August 2013 and August 2015 and who received resuscitative efforts by EMS. We compared clinical outcomes between day and night, defined as 7:00 pm-7:00 am based on EMS shift time including rates of sustained return of spontaneous circulation (ROSC), 30-day survival and favourable neurologic outcome (cerebral performance category 1 or 2). ALS quality measures included time to first medical contact, time to first shock, total dose of epinephrine, and multiple ALS performance measures. RESULTS: We included 1811 patients (37% female) with a mean age of 67 ± 16 years in our analyses. Rates of ROSC and 30-day survival with favourable neurological outcome did not differ between day or night (30% vs 28%, p =  0.33; 12% vs. 11%, p =  0.51, respectively). These results remained unchanged after multivariate adjustment for ROSC (RR, 1.1; 95% CI, 1.0-1.3, p = 0.19) and 30-day survival with favourable neurological outcome (RR, 1.2; 95% CI, 1.0-1.5, p =  0.10). The quality of ALS did not differ between day and night. CONCLUSIONS: In contrast to previous studies, there was no significant difference in sustained ROSC rates and 30-day survival with favourable neurological outcome after OHCA between day and night in the city of Vienna. This is likely due to nearly identical high bystander CPR rates and identical ALS performance provided by EMS personnel irrespective of time of the day.

Added value of the DIC score and of D-dimer to predict outcome after successfully resuscitated out-of-hospital cardiac arrest.

BACKGROUND: Recent Korean data suggest a high prevalence of overt disseminated intravascular coagulation (DIC) and a good predictive performance of the ISTH DIC score in successfully resuscitated out-of-hospital cardiac arrest. OBJECTIVES: We hypothesised that in a European cohort of resuscitated out-of-hospital cardiac arrest patients the prevalence of DIC is substantially lower. Furthermore, the determination of D-dimer levels at admission, but not the DIC score, could improve mortality prediction above traditional predictors. PATIENTS/METHODS: Data were extracted from a prospective cardiac arrest registry including patients admitted between 2006 and 2015, who achieved return of spontaneous circulation and had parameters for DIC score calculation available. The primary outcome was the prevalence of overt DIC at admission. Secondary outcomes included the association of overt DIC with 30-day mortality and the contribution of the DIC score and D-dimer levels to 30-day mortality prediction using logistic regression. Three stepwise models were evaluated by receiver-operating-characteristic analysis. RESULTS: Out of 1179 patients 388 were included in the study. Overt DIC was present in 8% of patients and associated with substantial 30-day mortality (83% vs. 39%). The AUC for model 1, including traditional mortality predictors, was 0.83. The inclusion of D-dimer levels significantly improved prognostication above traditional predictors (model 3, AUC 0.89), whereas the inclusion of the DIC Score had no effect on mortality prediction (model 2, AUC 0.83). CONCLUSION: Overt DIC was rare in a European cohort of out-of-hospital cardiac arrest patients. D-dimer levels improved 30-day mortality prediction and provided added value to assess early mortality risk after successful resuscitation.

Emergency extracorporeal life support and ongoing resuscitation: a retrospective comparison for refractory out-of-hospital cardiac arrest.

BACKGROUND: In refractory cardiac arrest, with cardiopulmonary resuscitation (CPR) for more than 30 min, chances of survival are small. Extracorporeal cardiopulmonary resuscitation (ECPR) is an option for certain patients with cardiac arrest. The aim of this study was to evaluate characteristics of patients selected for ECPR. METHODS: Anonymised data of adult patients suffering refractory cardiac arrest, transported with ongoing CPR to an ED of a tertiary care centre between 2002 and 2012 were analysed. Outcome measure was the selection for ECPR. Secondary outcome was 180 days survival in good neurological condition. RESULTS: Overall, 239 patients fulfilled the inclusion criteria. ECPR was initiated in seven patients. Patients treated with ECPR were younger (46 vs 60 years; p=0.04), had shorter intervals before CPR was started (0 vs 1 min; p=0.013), faster admissions at the ED (38 vs 56 min; p=0.31) and lower blood glucose levels on admission (14 vs 21 mmol/L; p=0.018). Survival to discharge in good neurological condition was achieved in 14 (6%) of all patients. One patient in the ECPR group survived in excellent neurological condition. Age was independently associated with the selection for ECPR (OR 0.07; 95% CI 0.01 to 0.85; p=0.037). CONCLUSIONS: Emergency extracorporeal life support was used for a highly selected group of patients in refractory cardiac arrest. Several parameters were associated with the decision, but only age was independently associated with the selection for ECPR. The patient selection resulting in a survival of one patient out of seven treated seems reasonable. Randomised controlled trials evaluating the age limit as selection criteria are urgently needed to confirm these findings.

Real-world use, effectiveness and safety of anti-viral treatment in chronic hepatitis C genotype 3 infection.

BACKGROUND: Treatment of chronic hepatitis C genotype 3 (GT3) is more challenging compared with other genotypes. Since 2014, several new treatment regimens have been approved but sometimes based on limited data. AIM: To validate the use, effectiveness and safety of anti-viral treatment in chronic hepatitis C genotype 3 infection under real-word conditions. METHODS: The German Hepatitis C-Registry is a large national non-interventional real-world study for patients with chronic hepatitis C. A total of 1322 GT3 patients were enrolled (211 untreated and 1111 treated patients). RESULTS: Between February 2014 and September 2015, five different treatment strategies have been used (PegIFN+RBV, PegIFN+RBV+SOF, SOF+RBV, DCV+SOF±RBV, LDV/SOF±RBV). Treatment uptake and use of treatment concepts changed markedly and rapidly during the study influenced by new approvals, guideline recommendations, and label updates. PegIFN-based therapies constantly declined while DCV-based therapies increased with one interruption after the approval of LDV/SOF, which was frequently used until new guidelines recommended not using this combination for GT3. Per-protocol SVR ranged from 80.9% in the PegIFN+RBV group to 96.1% in PegIFN+RBV+SOF treated patients. Treatment-experienced patients with cirrhosis showed a suboptimal SVR of 68% for SOF+RBV but a high SVR of 90-95% for DCV+SOF±RBV. The safety analysis showed more adverse events and a stronger decline of haemoglobin for RBV containing regimens. CONCLUSIONS: Real-world data can validate the effectiveness and safety for treatment regimens that had previously been approved with limited data, in particular for specific subgroups of patients. The present study demonstrates how rapid new scientific data, new treatment guidelines, new drug approvals and label changes are implemented into routine clinical practice today.

[Technical assist devices : Perspectives and new developments]. / Technische Hilfssysteme : Perspektiven und neue Entwicklungen.

The development of technical assist devices in the context of cardiopulmonary resuscitation (CPR) reaches back to the early roots of modern resuscitation research. This article covers the subjects of extracorporeal CPR (ECPR), including extracorporeal life support (ECLS), emergency ECLS (EECLS) and mechanical resuscitation devices. Specifically, the potential use of active compression-decompression CPR (ACD-CPR), impedance threshold devices (ITD) and capnography as additional measures during resuscitation are described in detail. Furthermore, the article presents a compact preview of the potential future developments of technical aids in the field of life support and postresuscitation care.

Cerebral and Peripheral Metabolism to Predict Successful Reperfusion After Cardiac Arrest in Rats: A Microdialysis Study.

BACKGROUND: In clinical practice, monitoring of the efficacy of resuscitation can be challenging. The prediction of cerebral and overall outcome in particular is an unmet medical need. Microdialysis is a minimally invasive technique for the continuous determination of metabolic parameters in vivo. Using this technique, we set out to establish a model allowing for concomitant determination of cerebral and peripheral metabolism in a cardiac arrest setting in rodents. METHODS: Microdialysis settings were optimized in vitro and then used in male Sprague-Dawley rats. Probes were implanted into the CA1 region of the right hippocampus and the right femoral vein. With a time interval of 8 min, glucose, lactate, pyruvate, and glutamate levels were determined during baseline conditions, untreated ventricular fibrillation cardiac arrest, cardiopulmonary resuscitation (CPR), reperfusion, and death. RESULTS: In 16 rodents, restoration of spontaneous circulation was achieved in seven animals. Characteristic metabolic changes were evident during cardiac arrest and reperfusion with both probes. Ischemic patterns in peripheral compartments were delayed and more variable compared to the changes in cerebral metabolism highlighting the importance of cerebral metabolic monitoring. Microdialysis allowed distinguishing between survivors and non-survivors 8 min after termination of CPR. Cerebral glutamate showed a trend toward higher levels in non-survivors during CPR. CONCLUSIONS: We established a new rodent model for research in hypoxic ischemic encephalopathy. This setting allows to investigate the impact of resuscitation methods on cerebral and peripheral metabolism simultaneously. The present model may be used to evaluate different resuscitation strategies in order to optimize brain survival in future studies.

Selective inhibition of HDAC8 decreases neuroblastoma growth in vitro and in vivo and enhances retinoic acid-mediated differentiation.

For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents.

MicroRNAs and the response to injury in atherosclerosis.

Endothelial cells (ECs) at arterial branching points are physiologically subjected to chronic damage by disturbed blood flow, which triggers a vascular wound healing response. Additional damage by hyperlipidaemia perturbs this delicate balance of endothelial injury and regeneration, and the progressive accumulation of noxious modified lipoproteins leads to macrophage death. Several miRNAs such as miR-92a and miR-712, which modulate EC proliferation and inflammation, are up-regulated by disturbed flow in ECs, and contribute to atherosclerosis. In addition, reduced endothelial levels of miR-126-5p limit the regenerative capacity of ECs, which becomes apparent by insufficient endothelial repair under hyperlipidemic stress. In macrophages, miR-342-5p induces the expression of miR-155 during the progression of atherosclerosis, which promotes inflammatory gene expression and inhibits efferocytosis by targeting Bcl6, thus contributing to necrotic core formation. Deciphering the complex cell- and context-specific effects of miRNAs during vascular wound healing appears essential for the development of miRNA-based therapies of atherosclerosis.

Cardiac arrest due to accidental hypothermia--a 20 year review of a rare condition in an urban area.

BACKGROUND: Accidental hypothermic cardiac arrest is associated with unfortunate prognosis and large studies are rare. We therefore have performed an outcome analysis in patients that were admitted to Vienna University Hospital with the diagnosis of accidental hypothermic cardiac arrest. METHODS: This study employed a retrospective outcome analysis of prospectively collected data in a selected cohort of hypothermic cardiac arrest patients. We screened 3800 cardiac arrest patients, treated at our department between 1991 and 2010, for eligibility. Inclusion criteria were cardiac arrest with a body core temperature ≤28 °C and return of spontaneous circulation. RESULTS: A total of 18 patients who achieved return of spontaneous circulation were analysed. Nine patients (50%) achieved survival in good neurologic condition (defined as cerebral performance category CPC 1 or 2). Accidental hypothermia with consecutive cardiac arrest was caused by intoxication in most cases (67%). These patients had a better outcome than patients with other causes of accidental hypothermic cardiac arrest (OR=28; 95%KI 2-37.9; p<0.01). Hypothermia associated typical ECG changes after return of spontaneous circulation (Osborne waves) were more frequent in the surviving population (OR 16; 95%KI 1.3-19.5; p=0.05). CONCLUSIONS: Accidental hypothermic cardiac arrest in a central European urban area is rare. Prognosis was excellent in patients where hypothermic cardiac arrest was caused by intoxication.

"Anatomy and imaging": 10 years of experience with an interdisciplinary teaching project in preclinical medical education - from an elective to a curricular course.

PURPOSE: Presentation of an interdisciplinary, interactive, tutor-based preclinical teaching project called "Anatomy and Imaging". MATERIALS AND METHODS: Experience report, analysis of evaluation results and selective literature review. RESULTS: From 2001 to 2012, 618 students took the basic course (4 periods per week throughout the semester) and 316 took the advanced course (2 periods per week). We reviewed 557 (return rate 90.1 %) and 292 (92.4 %) completed evaluation forms of the basic and the advanced course. Results showed overall high satisfaction with the courses (1.33 and 1.56, respectively, on a 5-point Likert scale). The recognizability of the relevance of the course content for medical training, the promotion of the interest in medicine and the quality of the student tutors were evaluated especially positively. CONCLUSION: The "Anatomy and Imaging" teaching project is a successful concept for integrating medical imaging into the preclinical stage of medical education. The course was offered as part of the curriculum in 2013 for the first time. "Anatomia in mortuis" and "Anatomia in vivo" are not regarded as rivaling entities in the delivery of knowledge, but as complementary methods.

Pain in adult patients with Pompe disease: a cross-sectional survey.

BACKGROUND: Pompe disease is a rare hereditary metabolic myopathy caused by a deficiency of acid-α-glucosidase. We investigated the presence and severity of pain and its interference with daily activities in a large group of adults with Pompe disease, who we compared with an age-matched control group. METHODS: Data were collected in a cross-sectional survey in Germany and The Netherlands. Pain was assessed using the short-form brief pain inventory (BPI). Patients also completed the Short Form-36 item (SF-36v2), the Hospital Anxiety and Depression Scale (HADS) and the Rotterdam Handicap Scale (RHS). RESULTS: Forty-five percent of the 124 adult Pompe patients reported having had pain in the previous 24h, against 27% of the 111 controls (p=0.004). The median pain severity score in Pompe patients reporting pain was 3.1 (on a scale from 0 to 10), indicating mild pain; against 2.6 amongst controls (p=0.06). The median score of pain interference with daily activities in patients who reported pain was 3.3, against 1.3 in controls (p=0.001). Relative to patients without pain, those with pain had lower RHS scores (p=0.02), lower SF-36 Physical and Mental component summary scores (p<0.001 and p=0.049), and higher levels of depression and anxiety (p=0.005 and p=0.003). CONCLUSIONS: To date, this is one of the largest studies on pain in a specific neuromuscular disorder. Nearly one in two Pompe patients had experienced pain in the previous 24h. Although pain severity and its interference with daily life were mild, pain was related to a reduced quality of life, less participation in daily life, and greater depression and anxiety. Its management should therefore be seen as part of clinical practice involving Pompe patients.

Screening for autoantibodies in chronic hepatitis C patients has no effect on treatment initiation or outcome.

Autoantibodies in hepatitis C virus-infected patients may indicate autoimmune hepatitis or other immune-mediated diseases. This may impact safety and efficacy of interferon-based therapy of chronic hepatitis C. We investigated the association between a positive test result for a variety of autoantibodies and the initiation and efficacy of therapy for chronic hepatitis C. We analysed an observational cohort of 24 306 patients for an association between autoantibodies and treatment outcome. 8241 patients were tested simultaneously for antinuclear antibodies (ANA), liver kidney microsomal antibodies (LKM), smooth muscle antibodies (SMA) and antimitochondrial antibodies (AMA). Matched-pair analysis was performed matching one autoantibody-positive patient to three controls. Control patients had negative tests for all four antibodies. Analyses were performed for patients with a single positive autoantibody test and for patients with multiple positive autoantibody tests. A positive test result for ANA, LKM, SMA or AMA did not affect the physician's decision to initiate therapy with pegylated interferon and ribavirin. In addition, a positive test for one or multiple autoantibodies did not adversely affect sustained virologic response. There was no difference in fibrosis stage or alanine transaminase at baseline or during therapy irrespective of antibody status. Thyroid dysfunction was more frequent in patients with positive LKM antibodies (P = 0.004). Initiation of therapy for chronic hepatitis C and outcome were not affected by the presence of ANA, LKM, SMA or AMA. Routine testing of these autoantibodies seems not warranted. Determination of autoantibodies should be guided by individualized clinical decisions.

Monitoring of unfractionated heparin with rotational thrombelastometry using the prothrombinase-induced clotting time reagent (PiCT®).

PURPOSE: To achieve sufficient and safe anticoagulation with unfractionated heparin (UFH) a close and reliable drug monitoring is necessary. In general, the activated partial thromboplastin time (APTT) is used for this purpose. In acute phase response, however, the APTT test procedure might be unreliable e.g. with false low results in the presence of elevated factor VIII. In this so called heparin resistance, measurement of anti-Xa activity is recommended over APTT to avoid potentially harmful dose escalation. A combination of anti-Xa measurement and global hemostatic testing with ROTEM® employing the anti-Xa sensitive PiCT® reagent showed high correlation with enoxaparin levels. This test modification could also be suitable for monitoring UFH. Aim of the study was to evaluate the correlation between PiCT®-ROTEM® and levels of UFH. METHODS: In this in-vitro study blood samples from healthy volunteers were spiked with UFH and subjected to different ROTEM® tests. RESULTS: There was a linear correlation between UFH level and clotting time (CT) in the PiCT®-ROTEM® test with an excellent correlation coefficient of 0.92. Additional endpoints showed similar results (PiCT®-ROTEM® MaxVel r = -0.85 and PiCT®-ROTEM® t_MaxVel r = 0.88). CONCLUSIONS: As a point-of-care applicable tool ROTEM® is immediately at hand. If further clinical studies confirm sensitivity in heparin resistance, PiCT®-ROTEM® could permit rapid UFH dose adjustments especially required in critical illness with acute phase response.

The role of microRNAs in arterial remodelling.

Adaptive alterations of the vessel wall architecture, called vascular remodelling, can be found in arterial hypertension, during the formation of aneurysms, in restenosis after vascular interventions, and in atherosclerosis. MicroRNAs (miR) critically affect the main cellular players in arterial remodelling and may either promote or inhibit the structural changes in the vessel wall. They regulate the phenotype of smooth muscle cells (SMCs) and control the inflammatory response in endothelial cells and macrophages. In SMCs, different sets of miRs induce either a synthetic or contractile phenotype, respectively. The conversion into a synthetic SMC phenotype is a crucial event in arterial remodelling. Therefore, reprogramming of the SMC phenotype by miR targeting can modulate the remodelling process. Furthermore, the effects of stimuli that induce remodelling, such as shear stress, angiotensin II, oxidised low-density lipoprotein, or apoptosis, on endothelial cells are mediated by miRs. The endothelial cell-specific miR-126, for example, is transferred in microvesicles from apoptotic endothelial cells and plays a protective role in atherogenesis. The inflammatory response of the innate immune system, especially through macrophages, promotes arterial remodelling. miR-155 induces the expression of inflammatory cytokines, whereas miR-146a and miR-147 are involved in the resolution phase of inflammation. However, in vivo data on the role of miRs in vascular remodelling are still scarce, which are required to test the therapeutic potential of the available, highly effective miR inhibitors.

Contractile activity of human skeletal muscle cells prevents insulin resistance by inhibiting pro-inflammatory signalling pathways.

AIMS/HYPOTHESIS: Obesity is closely associated with muscle insulin resistance and is a major risk factor for the pathogenesis of type 2 diabetes. Regular physical activity not only prevents obesity, but also considerably improves insulin sensitivity and skeletal muscle metabolism. We sought to establish and characterise an in vitro model of human skeletal muscle contraction, with a view to directly studying the signalling pathways and mechanisms that are involved in the beneficial effects of muscle activity. METHODS: Contracting human skeletal muscle cell cultures were established by applying electrical pulse stimulation. To induce insulin resistance, skeletal muscle cells were incubated with human adipocyte-derived conditioned medium, monocyte chemotactic protein (MCP)-1 and chemerin. RESULTS: Similarly to in exercising skeletal muscle in vivo, electrical pulse stimulation induced contractile activity in human skeletal muscle cells, combined with the formation of sarcomeres, activation of AMP-activated protein kinase (AMPK) and increased IL-6 secretion. Insulin-stimulated glucose uptake was substantially elevated in contracting cells compared with control. The incubation of skeletal muscle cells with adipocyte-conditioned media, chemerin and MCP-1 significantly reduced the insulin-stimulated phosphorylation of Akt. This effect was abrogated by concomitant pulse stimulation of the cells. Additionally, pro-inflammatory signalling by adipocyte-derived factors was completely prevented by electrical pulse stimulation of the myotubes. CONCLUSIONS/INTERPRETATION: We showed that the effects of electrical pulse stimulation on skeletal muscle cells were similar to the effect of exercise on skeletal muscle in vivo in terms of enhanced AMPK activation and IL-6 secretion. In our model, muscle contractile activity eliminates insulin resistance by blocking pro-inflammatory signalling pathways. This novel model therefore provides a unique tool for investigating the molecular mechanisms that mediate the beneficial effects of muscle contraction.

Pentobarbital inhibition of human recombinant alpha1A P/Q-type voltage-gated calcium channels involves slow, open channel block.

BACKGROUND AND PURPOSE: Pre-synaptic neurotransmitter release is largely dependent on Ca(2+) entry through P/Q-type (Ca(V)2.1) voltage-gated Ca(2+) channels (PQCCs) at most mammalian, central, fast synapses. Barbiturates are clinical depressants and inhibit pre-synaptic Ca(2+) entry. PQCC barbiturate pharmacology is generally unclear, specifically in man. The pharmacology of the barbiturate pentobarbital (PB) in human recombinant alpha(1A) PQCCs has been characterized. EXPERIMENTAL APPROACH: PB effects on macroscopic Ca(2+)(I(Ca)) and Ba(2+)(I(Ba)) currents were studied using whole-cell patch clamp recording in HEK-293 cells heterologously expressing (alpha(1A))(human)(beta(2a)alpha(2)delta-1)(rabbit) PQCCs. KEY RESULTS: PB reversibly depressed peak current (I(peak)) and enhanced apparent inactivation (fractional current at 800 ms, r(800)) in a concentration-dependent fashion irrespective of charge carrier (50% inhibitory concentration: I(peak), 656 microM; r(800), 104 microM). Rate of mono-exponential I(Ba) decay was linearly dependent on PB concentration. PB reduced channel availability by deepening non-steady-state inactivation curves without altering voltage dependence, slowed recovery from activity-induced unavailable states and produced use-dependent block. PB (100 microM) induced use-dependent block during physiological, high frequency pulse trains and overall depressed PQCC activity by two-fold. CONCLUSION AND IMPLICATIONS: The results support a PB pharmacological mechanism involving a modulated receptor with preferential slow, bimolecular, open channel block (K(d)= 15 microM). Clinical PB concentrations (<200 microM) inhibit PQCC during high frequency activation that reduces computed neurotransmitter release by 16-fold and is comparable to the magnitude of Ca(2+)-dependent facilitation, G-protein modulation and intrinsic inactivation that play critical roles in PQCC modulation underlying synaptic plasticity. The results are consistent with the hypothesis that PB inhibition of PQCCs contributes to central nervous system depression underlying anticonvulsant therapy and general anaesthesia.